The overall objective of this proposal is to understand regulating mechanism of UDP-Ga1NAc: polypeptide -Ga1NAc transferases involved in the initial step of O-linked glycosylation and biosynthesis of oncofetal gylcopeptide epitopes, and to pursue biological roles of these epitopes in cancer tissue. Three major projects dealing with -GalNAc transferases in normal and oncofetal tissues will be undertaken. 1) UDP-Ga1NAc:VTHPGY -GaINAc transferase, which creates oncofetal peptide epitope on normally-occurring VTHPGY sequence of human fibronectin in cancer and fetal tissues, will be purified to homogeneity, and its properties characterized. Limited proteolytic digestion will be applied to the purified protein. Cleaved peptides will be analyzed by SDS/PAGE and separated by size-exclusion or by reverse-phase HPLC. Amino acid analysis of separated peptides will also be performed. 2) UDP-GalNAc:polypeptide -GalNAc transferase from normal adult tissue will be purified, and its properties characterized. The general methods planned to characteri UDP-GalNAc:VTHPGY -GalNAc transferase will also be applied to this enzyme. 3) Antibodies to UDP-GalNAc:polypeptide -GalNAc transferases will be prepared. Once prepared, specific antibodies to -GalNAc transferases will be used to confirm the enzymatic nature of the purified protein by inhibition assay or immunoprecipitation of the activity and to compare the enzymes from different tissues. The possibility of modifier that changes the acceptor specificity of UDP-GalNAc: polypeptide -GalNAc transferase will also be studied. These studies will provide important insight into how the initial step of 0-linked glycosylation is regulated oncodevelopmentally, and will form a basis for future studies aimed at genetic expression of -Ga1NAc transferases.